Abstract (Project 2) Rather than use passive transfer of well differentiated adult immunoregulatory cells as a means to create transplant tolerance, we have discovered that a population CpG activated pro-B cells are extraordinarily potent, far more potent on a per cell basis than Tregs and far easier to prepare. Transfer of only 40,000, but not 90,000, induces permanent engraftment and probably tolerance in MHC incompatible islet and cardiac allograft mouse models. We have discovered that CpG induces expression TIM-1 a potential master switch for expression of an immunoregulatory module in mature Bregs (see Project 1/ Kuchroo). We also learned that ligation of TIM-1 by anti-TIM-1 mAb induces expression of IL-10, a member of the regulatory model discovered by Kuchroo. The stepwise contributions of CpG and activation of the TIM-1 pathway are examined through RNAseq and NanoString based analysis. To more precisely characterize the fate and phenotype, proliferative and differentiation of CpG activated pro-B cells, lineage-tracking methods are utilized throughout. We know that CpG activated pro-B cells proliferate and differentiate after transfer when introduced in optimal, not excessive, cell number. Depending upon environmental cues CpG activated pro-B cells can differentiate into a variety Bregs but CpG activated pro-B cells from RAG KO mice do not generate Bregs. In this project we will analyze the cellular basis of tolerance believing that CpG activated B regs and their progeny create an environment conducive to activation, expansion and retention of donor specific Tregs. The specific but multifaceted requirements for B cell differentiation, including the nature of the niche impairing expansion and differentiation of CpG-ProBs, in the acquisition of tolerance will be analyzed through use of a panel of carefully selected gene knockout mice. The role of T and B regs in the induction and maintenance of tolerance will be determined through time course protocols that selectively destroy Tregs or the mature progeny of CpG activate pro cells. The diversity of CpG-ProB progeny and their expression of a regulatory module that is IL-10 inclusive will be examined by single cell RNAseq of CpG-ProBs and their progeny at the transplant.